DE10236925A1 - Process for immobilizing molecules on surfaces - Google Patents

Abstract

A method for immobilizing molecules on surfaces is proposed, wherein a largely planar surface is coated with a polymer and the molecules are subsequently immobilized on the surface by means of the polymer.

Description

The present invention relates to a method for Immobilization of compounds, especially molecules, such as for example biomolecules, on surfaces or solid Carriers.

Chip packaging techniques in which multiple ICs (integrated circuit) are housed in a stand known in the art. Here, for. B. stacked-die- Structure often uses intermediate layers of polymers which is the connection of the stacked ICs produce and at the same time the possibly sensitive Help mechanically protect the top of the bottom chip. The Production of such layers is part of the process in some factories Series manufacturing process and in particular can Thickness of such layers in the range of a few µm and even below it can be set exactly.

For many biochemical and biotechnological applications it makes sense to attach biomolecules to solid supports immobilize. For bioconjugation to inorganic substrates typically uses cross linkers that connect between the inorganic layer and the biomolecules produce. For example, EP 1132739 B1 describes a method which is used in bioconjugation Bind molecules to inorganic substrates via cross-linkers, such cross-linkers can be silanes, for example. It is a linker system in EP 1132739 B1 proposed which for the detection and isolation of Biomolecules and as part of a sensor or biochip or can be used as a diagnostic tool. For example, in biochemical processes immobilized enzyme can be used several times. Furthermore, is the immobilization of enzymes and other biomolecules Key technology in the development of bio-compatible Implants.

A total of several methods are known, molecules - for example biomolecules - on surfaces of supports tie. In the field of immunology, for example Polystyrene surfaces like PolySorp and MaxiSorp are used to Binding or conjugating biomolecules to surfaces.

Passivated and known from EP 0646 038 to produce stabilized, porous carriers and for bioconjugation use. These carriers have a reversible high Sorption capacity, which is essentially not associated with a non-specific adsorption of molecules such. B. Proteins, polysaccharides or oligo- or polynucleotides.

A process for attaching biomolecules to surfaces using linker-like groups is described in DE 100 04 884 described. The process involves a contact of the polymer having the linker-like groups a source of hydroxide ions, which makes biomolecules such. B. Heparin, can be applied to substrate surfaces.

• a) wie viele Moleküle binden insgesamt auf einer definierten Oberfläche und
• b) wie viele Moleküle weisen nach dem Prozess der Bindung noch eine Aktivität auf.

Two essential questions are relevant for the specialist in the context of bioconjugation:

• a) how many molecules bind in total on a defined surface and
• b) how many molecules still show activity after the binding process.

A disadvantage of the known methods for bioconjugation is the linker used to attach the molecules to the Tie straps. The presence of the linkers sets the Activity of the bound molecules adversely. The well-known Procedures continue to be time consuming and laborious the linker or the corresponding analogues expensive.

The technical on which the present invention is based The problem is to provide a method that a simple and inexpensive conjugation or Immobilization of biomolecules on a surface enables and at largely the activity of the bound molecules preserved.

This goal is achieved through a process according to the characteristics of the Claim 1 solved. Advantageous embodiments of the Invention are the subject of the dependent claims.

In the inventive method for immobilizing Molecules on surfaces, is intended to be a layer of one hydrophobic and in particular non-swellable polymer to apply the surface and molecules on a surface immobilize the polymer layer.

Such hydrophobic polymers are, for example, polyimide or polystyrene. The surface on which the Polymer layer is applied, preferably consists of a inorganic material, such as a Semiconductor material, in particular silicon, a semiconductor oxide, especially silicon dioxide, glass, nitride or ceramic.

Have hydrophobic polymers such as polyimide or polystyrene the advantage of using conventional, in the Semiconductor technology known processes on the surface of a inorganic carrier can be applied. About that They also electrically isolate the carrier from those on the Surface of the polymer layer applied molecules or in Related substances. In for example made of a semiconductor material Carriers can thus electrical sensors and Evaluation circuits are integrated without their function by the the surface of the polymer layer applied molecules and To influence substances negatively.

The surface of the carrier can in the inventive Process completely or only partially with the organic hydrophobic polymer are covered, with the help of an in parts of the mask process common in the semiconductor industry Surface can be left out. This can later electrical connection contacts (bonds) on the carrier, For example, a chip can be attached. It is also possible to leave out parts of the surface for other reasons and so areas of the optionally inorganic surface to be left uncovered, or vice versa only certain ones Place the surface to be coated with the polymer to which molecules such as biomolecules later adhere can.

The polymer layer is used for the immobilization organic molecules that connect with this can be brought into contact. The contact is done in such a way that the molecules are bound site-specifically.

Sensor elements are preferably in the carrier below the Surface on which the polymer layer is applied, integrated to take measurements on the surface of the To be able to carry out polymer layer immobilized molecules. These measurements can be used, for example, to: Properties of the biomolecules or chemical reactions involved in characterize their surroundings.

For example, antibodies on surfaces can be more hydrophobic Polymer layers, for example made of a polyimide or a polystyrene with which serve as a carrier Semiconductor body or semiconductor layers are easily coated can be bound well, so that subsequently classic detection reactions such as B. Perform ELISA reactions to let.

Are molecules related to the present invention in particular peptides, proteins, genes and their fragments, Nucleic acids, carbohydrate structures like sugar, cells and their fragments, cell membrane components and / or hormones. Of course, microorganisms, cell extracts, Ligands, antigens, antibodies, receptors, lectins, Glycopeptides and / or lipids as a molecule on the surface of the Polymer layer can be immobilized. With the microorganisms can be living or dead microorganisms, the living microorganisms in the sense of the invention affect both growing and resting cells. The Microorganisms can be caused by intracellular cell cross-linking be pre-immobilized on the carrier, taking under Pre-immobilization means all procedures that are understood before Immobilize according to the inventive method to a Fixation of the molecules or cells can result. In which Inclusion in a polymer matrix for pre-immobilization of the Microorganisms are expediently biopolymers used, such as polysaccharides or proteins or but synthetic polymers.

Furthermore, it is of course also possible to use ligands as molecules on the surface of the polymer layer immobilize. Ligands in the sense of the invention are e.g. B. Molecules, such as proteins or ions, around a Central structure can be grouped. Ligands can be a and be multi-toothed. Molecules can also be used as ligands understood at specific points of Macromolecules are bound, for example substrates or coenzymes of a protein. For the purposes of the invention are among molecules or biomolecules also antigens and / or antibodies understand. Antigens in the sense of the invention are all substances that can trigger an immune response. It can be foreign, natural or synthetic macromolecules, especially proteins or polysaccharides, with a Molecular weight of over 2 kilo daltons are trading as well Surface structures of foreign particles. An antigen according to the Invention can consist of a high molecular weight part, which as Serves substrate of mostly several low molecular weight groups, responsible for the specificity of the immune response and the response of the Antigens with the corresponding immunoglobulins are decisive. The antigens can be polyvalent and monovalent be and so with one or more types of antibodies interact.

It can also be provided instead of the antigens antibodies immobilize on the carrier. Being under antibodies especially understood glycoproteins that specifically with interact with an antigen. It comes through the interaction for the formation of antigen-antibody complexes. The antibodies can, for example, different groups of immunoglobulins his. The antibodies can be used as intact antibodies or as various fragments, for example by means of cleavage can be generated by different peptidases, be immobilized. The antibodies can be used before or during or modified after immobilization on the support, for example by reduction, oxidation or by a Oligomerization. Furthermore, it is also possible as biomolecules Use receptors. Examples are receptors Proteins with an extracellular signaling molecule, for example a ligand, interact and through Conformation changes certain functions, especially about secondary messengers, activate or initiate. receptors In the sense of the invention, however, special cells can also be used be the stimuli and the appropriate information hand off; Examples of this would be photo, chemical, thermal and baroreceptors.

Preferably, the surface on which the Polymer layer is applied, largely planar, d. i.e. it is a surface with low roughness such as B. Surfaces of semiconductor layers or semiconductor bodies with integrated circuits (IC surfaces), however local microscopic structures, e.g. B. to accommodate Biomolecules would be suitable.

Under immobilization or pre-immobilization in the sense of Invention are all methods of restricting the Mobility and solubility of molecules on chemical, to understand biological and / or physical ways, the Pre-immobilization all procedures for fixing the molecules concerns that before the immobilization according to the method according to the invention are carried out. Immobilization and / or pre-immobilization can be different Methods are done like binding the molecules to each other or on carriers, by sticking to the network of a polymer Matrix or enclosed by membranes. Through the Immobilization not only makes the molecules reusable, but after the process of interacting with the sample can be easily separated again. You can be in a lot higher local concentrations and in continuous Use flow systems. The bond or the Immobilization of the molecules on the carrier can be done by direct Carrier connection and by cross-linking. The Carrier binding takes place according to the invention in particular by ionic, adsorptive or by covalent binding. The cross-linking In the sense of the invention, the molecules are crosslinked with each other or with other polymers. In the The molecules are immobilized by inclusion in gel structures or enclosed in membranes before they are on the Surface of the carrier can be immobilized.

Numerous possibilities are known to the person skilled in the art which Immobilize molecules on the carrier. Immobilization should be done so that each probe or molecule a defined position on the carrier can be assigned and that each position on the carrier is evaluated independently can be. However, it may also be desirable that the Places of application of different molecules or probes partially or completely overlap or that biomolecule mixtures be applied. The immobilization can, for example, with a process based on semiconductor technology respectively. Essentially, the molecules or biomolecules can two basically different ways on the carrier be immobilized: (a) on the one hand is the in situ synthesis of the Molecules at defined positions on the carrier successive coupling of monomeric synthesis building blocks possible, (b) to another is filing and immobilizing from before synthesized or library derived biomolecules or other molecules at defined positions in particular functionalized carrier material possible. For this you can both spotting and printing processes are used. Spotting means processes in which Drops of liquid in which the molecules are located on the Carriers are deposited, whereby through surface interaction and drying essentially round spots arise. But other printing processes also make it possible to insert the molecules in place defined areas on the surface of the carrier, which ensures stable binding of the samples to the Substrate surface of the molecules with high coupling efficiency can. All immobilization measures known to the person skilled in the art of biomolecules on z. B. Column materials can also be used to attach the molecules to the carrier immobilize.

Selected methods of immobilization or Pre-immobilization is, for example, contact tip printing, ring and Pin Printing, Nanoelectric Printing and Nanopipetting, Bubble Jet Printing, TopSpot Printing, Micro Contact Printing, Micro Fluidic Networks Methods, Photolithographic Activation- Process, photoresist lithography, electrochemical focusing and Micro Wet Printing. According to the invention, all of these can Procedures are applied.

For the purposes of the invention, inorganic supports can be used Surfaces comprising metal, polypropylene, teflon, polyethylene, Polyester, polystyrene, nitride, ceramic and / or glass are used, or IC (integrated circuit) Surfaces, silicon, silicon dioxide or others. Metals in All compounds whose A crystal lattice creates cohesion. The border between Metals and non-metals is fluent, so that too Elements Ce, Sn, As and Sb in the sense of the invention metals are. The metals according to the invention also include metallic glasses, that is, materials that are in one metastable, largely amorphous state. Metallically conductive polymers are of course also metals in the sense of the invention. Metals advantageously have in According to the invention, in particular good strength, a good hardness and wear resistance, high toughness and good electrical and thermal conductivity. Polypropylenes in the sense of the invention are thermoplastic Polymers of propylene. Polypropylenes stand out especially due to high hardness, resilience, rigidity and Heat resistance. Teflon according to the invention Polytetrafluoroethylene, which is advantageously good have thermoplastic properties. Polyethylenes are created in particular by a polymerization of ethylene by im essentially two different methods, the high pressure and the low pressure process. Polyethylenes used in High-pressure processes are advantageously produced low density. The properties of carriers that Polypropylene are essentially covered by the Character of polyethylene as partially crystalline Determined hydrocarbon. Polyethylenes are advantageously up to 60 ° C practical in all common solvents insoluble. Polar liquids such as Almost no alcohol, esters and ketones at room temperature Swelling of polyethylenes and thus the carrier coating. Against water, alkalis and salt solutions as well as inorganic Acids advantageously behave completely in polyethylenes indifferent. Carriers comprising polyethylenes have e.g. B. a very low water vapor permeability. The carrier can however expediently also include polyester. polyester for the purposes of the invention are compounds which are Ring opening polymerization of lactones or by polycondensation of hydroxycarboxylic acids or of diols and dicarboxylic acids or dicarboxylic acid derivatives. Polyester in According to the invention, polyester resins also include Polyester imides, polyester rubbers, polyester polyols and Polyesterpolyuretane. Polyesters are advantageously thermoplastics and have a distinct material character. they draw are characterized, for example, by high thermal stability and can lead to alloys with metals, such as Copper, aluminum and magnesium can be processed.

However, it can also be provided that the carrier is ceramic includes. Ceramic in the sense of the invention is one Collective name for especially inorganic and predominantly non-metallic compounds containing more than 30% by volume include crystalline materials. The skilled person is different Ceramics or ceramic materials known that he as Carrier can use. For example, it can be so-called earthenware, split plates, laboratory porcelain, Alumina ceramics, permanent magnet materials, silica stones and Act magnesia stones. In the case of clay ceramic materials, In terms of the invention in coarse and fine materials distinguished, whereby fine clay-ceramic materials earthenware, earthenware, Stoneware and porcelain include. However, you can use Advantage also special ceramic materials such as glass, oxide ceramics, SiC stones and molten cast stones as supports be used. The carrier can preferably also be glass include. Glass in the sense of the invention are substances, in the amorphous, non-crystalline solid state, that is, the glass state can be considered frozen in the sense of the invention Collect supercooled liquid or melt. Glasses are therefore inorganic or organic, mostly oxidic melt products, by an insertion process without crystallization of the Melt phase components converted into a solid state were. Of course, within the meaning of the invention Crystals, melts and supercooled melts as glasses specific. The glasses can be flat glass, Laboratory equipment glass, lead crystal glass, fiber glass, optical glass fibers and be others. Of course it is also possible that Silicate-free glasses, for example phosphate glasses, are used become. However, the carrier can also be such that optical glasses, that is, e.g. B. glasses with special optical refractive indexes can be used.

It is self-evident in the sense of the invention that the surface of the carrier can be modified. The carriers can be modified in particular by the action of biological, physical and / or chemical influences. Physical action would be, for example, polishing, etching, pickling, sandblasting, but also physical processes that lead to hardening, coating, tempering, covering with protective skins and the like. A surface treatment by biological action can include, for example, overgrowth by microorganisms. A chemical modification of the surface of the carrier includes, for example, treatment with acids, bases, metal oxides and others. The surface of the carrier can be modified such that the molecules adhere particularly well to the carrier or so that their activity is not disadvantageously modified. The surface modification also includes coating with poly-L-lysines, aminosilanes, aldehyde silanes, epoxy groups, gold, streptavidin, reactive groups, polyacrylamide pads, immobilized nitrocellulose and / or activated aldehydes or agarose aldehyde groups, which in particular binds are: DNA, COO ^- groups, NH ₂ groups, biotin, thiol groups and others. A surface modification of the carrier naturally also includes a treatment which leads to increased stability and breaking strength. Of course, especially when immobilizing biomolecules, classic surface modifications from histology can also be carried out.

In one embodiment of the invention is provided in certain portions of the surface of the polymer layer further semiconductor bodies or semiconductor layers with integrated Apply circuits or additional microsystems. As Polymer layer is particularly suitable here a polyimide, that for such applications in semiconductor technology is known. Polyimides are special high temperature resistant polymers; they advantageously have excellent mechanical, thermal and electrical properties. So far known applications of the polyimide in the Semiconductor technology particularly include buffer layers, Passivation layers, bonding layers and dielectric interlayers on the carrier. In particular, polyimides become liquid applied and then cured. In this curing step the polyimide advantageously receives the desired ones Characteristics. For the applications, the polyimide can be lithographically be structured. Of course, polyimide can also as an adhesion promoter for potting material and as Buffer layer can be used. The polyimide layer is reduced for example the stress in silicon caused by the encapsulation is caused and prevents cracks on the edges. The Polyimide in particular needs to be very even Temperature conditions are cured to crack in the polyimide and To prevent color irregularities. Low Oxygen values are e.g. B. advantageous to good adhesion to reach.

This also as a polymer layer for the immobilization of Polystyrene which can be used according to the invention is a thermoplastic, which is mainly due to radical polymerization of styrene is obtained. The radical end of a growing polymer chain never affects one Double bond in the ring because the benzene ring is an extraordinary is stable structure. This has several advantages when using polystyrene, for example Polystyrene resistant to acids, bases and alcohol.

In a further advantageous embodiment of the Invention, the hydrophobic polymer is only predefined in Areas applied to the surface.

In a further particularly preferred embodiment the surface becomes positive and / or by plasma treatment negatively charged electrically, d. that is, the surfaces are on the different places loaded differently. polymers In particular, materials come in various forms. The individual forms have different requirements to the machining process. Depending on the shape of the surface is this z. B. the plasmas in different ways accessible. Plasma treatment of the polymer surface can advantageously greatly increase the surface energy and enable other processing methods. At a Plasma treatment mainly react to the ions and radicals of the Plasmas with the polymer surface and create functional ones there Groups that have the surface properties of the polymer Determine advantage. By the positive or negative charge will in particular better wettability and / or better binding of the biomolecules achieved.

In a further preferred embodiment of the UV reactive molecules are invented by irradiation with UV Light covalently immobilized. For example, it can be provided, photolabile protecting groups on glass by light that selectively shines through a photolithographic mask, activate locally for oligosynthesis. The glass will then with photolabile molecules, such as DNA bases, flooded to the defined previously illuminated Tie array locations. For the next oligo bases in the sequences then other photolithographic masks used and the process repeated. For each base in the sample Oligo (per position) therefore 4 masks are required. With Advantages can be made directly from known ones Sequence databases take place, whereby a uniform standardization is achieved becomes.

Become hydrophobic molecules, especially biomolecules one of the above methods, for example one Printing process, on the surface of the hydrophobic Applied polymer layer, these molecules adhere due to a well-known interaction on this surface.

For the immobilization of molecules on the surface of the Polymer layer is in one embodiment of the inventive method provided the surface of the polymer layer at least in sections, for example using a conventional mask technique, in an oxygen plasma to activate. This will make the surface of the Polymer layer aldehyde groups, carboxy groups or hydroxide groups educated. These groups are hydrophilic and allow covalent bonds with biomolecules that activated them Areas, for example by printing with a die Solution containing molecules are applied. This covalent bonds are so stable that the polymer layer with the then immobilized molecules in soap can be cooked without breaking the bonds.

The surface is preferably only island-like Oxygen plasma treatment activated, which is the "island" surrounding areas of the polymer layer which have remained hydrophobic the applied solution runs on the surface limit.

In the following, the invention will be described with reference to Embodiments are described in more detail without referring to these examples to be limited.

example 1

Silicon sensor chips with CMOS photodiodes are used Polystyrene in a spin coater with a layer of approx. 100 to 200 nm Polystyrene covered. For this purpose, the chips with one 200 µl 0.1% (w / v) polystyrene solution in toluene for one minute 3000 rpm coated in a spin coater. Then the Sensor areas (photodiodes) with a protein solution in one printed grid-like arrangement. There are antibodies in PBS buffer used. The antibodies are each in a Concentration of 5 µg / ml used. Part of the grid is printed with antibodies using fluorescent dyes are conjugated. The antibodies are in a damp Chamber incubated at 4 ° C overnight and the unbound then rinsed with PBS buffer. After washing with Aqua dest. the success of immobilization with the help of a Fluorescence meter checked. The successful bond the antibody to the sensor areas is replaced by the Fluorescence of the antibodies detected. Then the chip sealed by attaching a PMMA reaction chamber. The reaction chamber is attached by connection of the PMMA to the polystyrene layer. The finished construction will yet through the use of a commercially available Stabilizing reagent for proteins is stabilized and ready to use.

Example 2

Silicon sensor chips with CMOS photodiodes are already on coated the waver with a 5 µm layer of polyimide. Then the polyimide is made with a copolymer Benzophenone methacrylate and acrylic acid coated. The bearers can then be easily combined with biomolecules such as DNA (5 µM Oligonucleotide in PBS buffer) are printed. The Immobilization is carried out by UV exposure at 300 nm for approx. 10 Minutes. The benzophenone of the copolymer forms radicals, which is covalent to the polyimide coating and to the DNA Create a bond. The same process can also be done with all other biomolecules such as proteins, especially Antibodies, peptides, sugars, lipids and triglycerides as well also perform complex structures of the same.

Claims (7)

1. A method for immobilizing molecules on surfaces, comprising the following process steps: Applying a layer of a hydrophobic polymer to the surface, - Immobilizing molecules on a surface of the layer. 2. The method of claim 1, wherein the polymer Is polyimide and / or polystyrene. 3. The method according to claim 1 or 2, wherein the Polymer layer on the surface only in predefined areas is applied. 4. The method according to any one of the preceding claims, in which the surface of the polymer layer at least in sections positive or negative electrical by plasma treatment is loaded. 5. The method according to any one of the preceding claims, at the UV reactive molecules by exposure to UV light covalently immobilized. 6. The method according to any one of the preceding claims, in which the polymer layer at least in sections in one Oxygen plasma is activated. 7. The method according to any one of the preceding claims, at which is part of the surface of the polymer layer for one Application with an integrated circuit (integrated circuit (IC)) or a microsystem.